靶向突变K-ras基因的小干扰RNA体内外抑制肺癌细胞生长的研究

目的探讨应用小于扰RNA（siRNA）敲除突变K-ms基因对肺癌细胞H441体内外生长的抑制作用。方法构建真核表达载体pSilencer3．1-K-ras^v12,转染H441细胞后应用半定量RT-PCR及Westernblotting检测突变K-l＇as基因mRNA及蛋白质的表达变化,噻唑蓝法检测H441细胞增殖速度的变化,流式细胞仪检测H441细胞凋亡率的变化。裸鼠皮下移植pSilencer3．1-K-ras^v12。处理的H441细胞,观察其成瘤性的改变。结果测序证实pSilencer3．1-K-ras^v12。真核表达载体构建成功。RT-PCR灰度比值结果示空载体组、阴性对照组、实验组K-rasmRNA相对表达量分别为：93．7％±2．2％、95．1％±2．5％、43．6％±3．1％,差异有统计学意义（F=78,P〈0．01）;Westernblotting结果示空载体组、阴性对照组、实验组K-ms蛋白相对表达量分别为：98．1％±2．4％、97．5％±2．0％、33．5％±3．7％,差异有统计学意义（F=93,P〈0．01）;经pSilencer3．1-K-ras^v12。作用的H441细胞生长受到明显抑制（P〈0．05）,凋亡率较对照组明显升高（F=8．9,P〈0．01）,H441细胞在裸鼠体内生长受到明显抑制。结论靶向突变K-ras基因的siRNA可以抑制肺癌H441细胞在体内外的生长速度,诱导细胞凋亡,为肺癌的基因治疗提供了新的思路和方法。

Study of growth inhibition of lung cancer cells by siRNA targeting mutant K-ras gene in vitro and in vivo

OBJECTIVE: To study the inhibitory effect of mutant K-ras gene depletion by small interfering RNA on the growth of lung cancer cell line-H441 cells in vitro and in vivo. METHODS: One pair of 63 bp reverse repeated sequence targeting mutant K-ras(V12) mRNA spaced by 9 bp nucleotide were synthesized and inserted into plasmid pSilencer3.1 eukaryotic expression vector. After transient and stable transfection into H441 cells, the mutant K-ras mRNA and protein level were measured by RT-PCR and Western blotting, and the H441 cells proliferation was measured by MTT method, and the apoptosis rate was detected by flow-cytometry. H441 cells treated with pSilencer3.1-K-ras(V12) were transplanted subcutaneously in nude mice and their tumorigenesis ability was observed. RESULTS: The recombinant plasmid pSilencer3.1-K-ras(V12) was successfully constructed by sequencing. The introduction of pSilencer3.1-K-ras(V12) was showed to efficiently and specifically inhibit the expression of K-ras(V12) gene according to the results of RT-PCR and Western blotting (P < 0.01, as compared with controls). The inhibitory effect on cell proliferation was confirmed by MTT test (P < 0.05, as compared with controls). Apoptosis rate of H441 cells treated with pSilencer3.1-K-ras(V12) was significantly higher than that of the control cells (P < 0.01). The test in vivo showed that downregulation of K-ras(V12) expression in H441 cells apparently affected their ability to form tumors in nude mice. CONCLUSIONS: siRNA targeting mutant K-ras mRNA can specifically suppress the expression of mutant K-ras gene in H441 cells, and therefore has a substantially inhibitory effect on cell proliferation in vitro and in vivo, it provides a new method and material to the gene therapy of lung cancer.